PROJECT SUMMARY Cell migration is critical to development and disease, and yet yet our understanding for this complex dynamic event is rather limited. Cell culture studies have uncovered crucial regulators of actin cytoskeleton, including the Rho family of GTPases (RhoA, Rac1, and Cdc42), that influence particular processes of cell migration. However, in vivo activities of these regulators and how they coordinate to promote efficient cell migration are not understood in detail. The long-term goal is to understand the molecular mechanisms that coordinate directed cell migration. To achieve this goal, the overall objective of this proposal is to determine how Cdc42ep1, an effector protein for Cdc42, interacts with other actin regulators to coordinate neural crest cell migration. Recent study from the lab revealed that Cdc42ep1 is essential in directed migration of neural crest cells during Xenopus embryogenesis. Using this in vivo cell migration system, two subcellular pools of Cdc42ep1 were revealed, one at the protrusive front and the other at the cell body and rear. These two pools of Cdc42ep1 interacts with Cdc42 and septin filaments, respectively, and these interactions can influence the balance of Cdc42ep1 between the two cytoplasmic pools. Therefore, the central hypothesis for this proposal is that Cdc42ep1 coordinates Cdc42- mediated membrane protrusion at the leading edge and the septin-actin cytoskeleton organization at the trailing edge to promote directed neural crest cell migration. The central hypothesis will be tested by pursuing the following three specific aims: 1) Determine the feedback regulation between Cdc42 and Cdc42ep1 and its impact on polarized actin dynamics and directed neural crest cell migration; 2) Determine the mechanisms of cooperation between Cdc42ep1 and septin filaments in controlling cell polarity and directional migration of neural crest cells; and 3) Determine the function and mechanism of septin filaments in regulating the formation and contractility of actin stress fibers. This work is a close collaboration with Tsygankov lab, where a morphodynamic cell migration model will be developed to test various molecular mechanisms and guide further experimental designs. By tightly integrating the in vivo and in silico experiments in a quantitative manner, the proposed research will uncover the mechanisms of how Cdc42ep1 integrates activities of Cdc42 and septin to organize actin dynamics at the protrusive front and the retractive rear to promote neural crest cell migration. This study will fill the knowledge gap of how local cytoskeletal arrangements are coordinated in directed cell migration. This knowledge is not limited to neural crest migration, but can be applied to other migration processes to provide a mechanistic understanding of in vivo cell migration in general. Therefore, the study will not only be critical for understanding the development of neural crest related birth defects, but also help improve our understanding of numerous human diseases that involve dysregulated cell migration in other contexts.